Oxygen free radicals and oxygen intermediates, especially singlet oxygen and the hydroxyl radical, cause extensive tissue damage. Free radicals and oxygen intermediates within living cells arise from endogenous sources, for example, from mitochondrial electron transport chain, oxidant enzymes, phagocytic cells and auto-oxidation reactions, as well as from exogenous sources such as cigarette smoke, "redox cycling" drugs and pesticides, heat stress, and ionizing radiation. These oxygen species damage compounds of all biochemical classes, including nucleic acids, protein and free amino acids, lipids and lipoproteins, carbohydrates, and connective tissue macromolecules. In addition, these oxygen species are believed to have an impact on cellular activities, such as membrane function, metabolism, and gene expression. Both singlet oxygen and hydroxyl radical are known to produce damaging strand breaks in DNA during reperfusion (specifically, during the reoxygenation aspect of reperfusion following ischemia) and during severe inflammatory conditions. Also, these oxidants, in the presence of metal ions (e.g., iron), initiate lipid peroxidation, which, in turn, produces mutagens, carcinogens, and other reactive oxygen species.
Numerous clinical conditions implicate oxygen radicals as the cause of tissue damage in single organs such as erythrocytes (e.g., lead poisoning), lungs (e.g., acute respiratory distress syndrome), heart and cardiovascular system (e.g., atherosclerosis), kidney (aminoglycoside nephrotoxicity). GI tract (e.g., free-fatty-acid-induced pancreatitis), brain and CNS (e.g., senile dimenti, hypertensive cerebrovascular injury, and cerebral trauma), eye (e.g., cataractogenesis), and skin (e.g., solar radiation and contact dermatitis.) Clinical conditions involving multiorgan disorders linked to oxygen radicals include, for example, inflammatory-immune injury, ischemiare-flow states, radiation injury, aging, cancer and amyloid diseases.
In spite of the more recently known deleterious effects of these oxygen species, the scientific community has focused its attention on the superoxide anion for the past two decades, leaving largely unexplored the role of singlet oxygen and the hydroxyl radical in human disease. Thus, no drugs that specifically target these oxygen species are currently available. It is one of the goals of the present invention to design a series of drugs that can scavenge or neutralize singlet oxygen and the hydroxyl radical in tissue experiencing oxidative stress. In particular, the need for such drugs is particularly great for protecting or treating cardiac tissue undergoing heart attack, angioplasty, and cardiac surgery. The invention also addresses the need for ameliorative drug therapies for ischemic stroke, vasospasm during subarachnoid hemorrhage, head injury, spinal cord injury, and neurosurgery, whereby the effects of free radical damage from reperfusion and inflammation would be prevented or lessened.
Another aspect of the invention is the control of tissue damage caused by the presence of cytokines and growth factors during severe inflammatory conditions (a.k.a. runaway inflammatory conditions) and by chemotactic cascades caused by surgical intervention, infectious diseases, parasitic and other inflammatory conditions. It is believed that cytokines are responsible for "communication" between cells which ultimately leads to gene activation. These discrete signaling molecules are considered benign to tissue in a healthy state. Cytokines include interleukins, various growth factors, interferons, and colony-stimulating factors. However, the net biological effect of the interaction of this class of molecules can also be inflammation and in this instance, cytokines are also known to play a major role in a wide variety of disease states such as cancer, allergy, infection, inflammation, angiogenesis, and differentiation. Cytokines and growth factors together are also believed to play a major role in restenosis (neointimal hyperplasia or proliferation following percutaneous transluminal coronary angioplasty and related procedures for removing blockages within blood vessels and lymph ducts).
The mechanism by which tissue is damaged during a runaway inflammatory response caused, for example, by a bacterial infection is as follows. Inflammatory cytokines such as tumor necrosis factor and interleukin-1 activate intracellular, microbicidal neutrophils. In their normal, defensive operation, neutrophils aggregate and release toxic granule proteins and the products of neutrophil oxidative burst to destroy the harmful microbe. However, in a runaway process, the neutrophil aggregation and release of microbicidal substances are not localized in the vicinity of the microbe, leading to damage of the host tissue. This chain of events occurs in a number of diseases associated with, e.g., infectious agents the immune system, chronic inflammation, and the respiratory system, as well as during numerous surgical procedures.
Accordingly it is another goal of the invention to design a series of drugs that can ameliorate tissue damaged by severe inflammatory responses, without interfering with host tissue defense mechanisms. In particular, the invention recognizes the great need for ameliorative drug therapies for stroke, heart attack, restenosis, adult respiratory distress syndrome (ARDS) and septic shock (the latter two conditions having a high likelihood of fatality), asthma, hearing loss associated with bacterial meningitis, inflammatory bowel conditions, and dozens of other conditions.
Blood flow reductions in the heart can result in dysfunction of this organ and cell death if the flow reduction is severe enough. Restoration of coronary blood flow early during a heart attack is becoming a clinical reality with the advent and improvements in thrombolytic, mechanical, and surgical interventions. While early restoration of blood flow by thrombolysis or following transient ischemia can prevent or mitigate the degree of cell death (infarction), reperfusion can still result in some degree of cardiac dysfunction or cell death (also referred to as stunned myocardia). Thus, it would be of great clinical value to find a means to preserve normal function of the heart during reperfusion and during various forms of cardiac surgery.
Additionally, heart disease is the biggest cause of death in the Western world. There are many different forms of heart disease and disease states can develop from a number of different factors including stress, diet, tobacco use, and genetic make up of the individual. Ischemia is a heart disease condition characterized as a local hypoxia caused by mechanical obstruction or occlusion of the blood supply. Oxygen radicals have been implicated as important mediators of tissue injury during myocardial ischemia and reperfusion. A number of studies have shown that free radicals, particularly superoxide anions (O.sub.2-) and hydroxyl radicals are generated following reperfusion of the ischemic myocardium and have linked the free radical generation to the loss of contractile function. Superoxide anion is relatively unreactive and is considered dangerous because its dismutation results in the formation of hydrogen peroxide which can potentially generate the highly reactive hydroxyl radical (OH) in the presence of transition metal ions. It is therefore generally believed that ultimate tissue damage occurs due to OH radicals. Indirect proof for the involvement of OH radicals in ischemia/reperfusion injury is derived from observations of a protective effect of OH radical scavengers such as dimethylthiourea (DMTU), dimethylsulfoxide, and mannitol. In addition, certain agents which prevent the formation of hydroxyl radicals have also demonstrated a protective effect, including deferoxamine, superoxide dismutase, and catalase.
Another active oxygen species is singlet molecular oxygen (.sup.1 O.sub.2). Singlet oxygen is not a radical; rather, it is an electronically excited state of oxygen which results from the promotion of an electron to higher energy orbitals. In Kukreja et al., Biochim. Biophys. A, 990:198-205 (1990), and Kukreja et al., Am. J. Physiol., 259:H1330-H1336 (1989), data was presented which demonstrated that superoxide anion or hydrogen peroxide are the least reactive species in damaging sarcolemma or sarooplasmic reticulum. Therefore, it might be inferred that the only species believed to be injurious in myocardial tissue is OH radical can initiate lipid peroxidation which can produce lipid free radicals that may become important sources of singlet oxygen in vivo. Hence, the damage often attributed to the OH radical could be the resultant effects of other intermediate reaction products, including lipid free radicals and singlet oxygen.
Janero et al., J. Mol. Cell Cardiol., 21:1111-1124 (1989), showed that .alpha.-tocopherol provides cellular protection by acting as a chain breaker in the lipid peroxidation process, not by scavenging the O.sub.2 -- radical per se. Singlet oxygen is also acted upon by .alpha.-tocopherol. Hearse et al., Circ. Res., 65:146-153 (1989), and Vandeplassche et al., J. Mol. Cell Cardiol., 22:287-301 (1990) (abstract) showed that .sup.1 O.sub.2 generated from exogenous sources is able to mimic ischemia/reperfusion induced myocardial damage. Tarr et al., J. Mol. Cell Cardiol., 21:539-543 (1989), recently reported that rose bengal, when applied extracellularly to frog atrial myocytes, induced a prolongation followed by a reduction of action potential duration. In addition, Donck et al., J. Mol. Cell Cardiol. 20:811-823 (1988) reported that isolated myocytes exposed to rose bengal light rapidly experience ultrastructural injury.
In Kukreja et al., Abs. of 63rd Sci. Sess. (AHA) (Dallas), 1068 (1990), it was reported that singlet oxygen generated from photosensitization of rose bengal induced significant inhibition of calcium uptake and Ca.sup.2+ --ATPase activity in isolated sarcoplasmic reticulum. This damage caused by singlet oxygen could be significantly reduced using L-histidine, but not with SOD or catalase. Misra et al., J. Biol. Chem., 265-15371-15374 (1990), reported that L-histidine is a scavenger of singlet oxygen. In contrast, SOD and catalase are scavengers of superoxide anion. Kim et al., Am. J. Physiol., 252:H252-H257 (1987), demonstrated that L-histidine provides significant protection of sarcolemmal Na.sup.+ K.sup.+ --ATPase activity following ischemia/reperfusion in guinea pig hearts.
Electrocardiography is a well known technique for examining the condition of the heart. There are four chambers in the human heart. In operation, the right atrium receives venous blood from the body and pumps it into the right ventricle which pumps the blood through the pulmonary network where the blood becomes oxygenated by the lungs. The oxygenated blood is returned to the left atrium and is pumped into the left ventricle. The left ventricle is the most powerful chamber of the heart and serves the function of propelling the blood throughout the body network. Typically, 2,000 gallons of blood a day are pumped through the heart of a normal individual, and the heart keeps this pace throughout the life of the individual (e.g., seventy years or more). An electrocardiograph apparatus enables doctors to monitor electrical changes in the heart muscle. All the functions of the body are motivated by a complex electromechanical system which is controlled through the brain and central nervous system. Each cell within the body is surrounded by a membrane which is electrically "polarized", meaning they each have positive and negative ions on opposite sides of the membrane. Contraction of a heart muscle cell causes an electrical current flow due to the positive and negative ions. Because all of the heart cells are intimately connected, the head organ acts as one very large cell. In the resting state (diastole), no current flows; however, as the heart expands and contracts, electrical current flows and can be sensed by electrodes.
Electrocardiography is the process of sensing and analyzing the current flow in the heart of a patient. Because the principal current detectable when a patient is at rest is produced by the heart, electrodes need not be connected directly to the heart. Typically, six or more electrodes are positioned on different portions of a patient's chest to sense the electric signals from the heart. The sensed signals are recorded on a monitor or strip chart and are referred to as an electrocardiogram. The electrocardiogram is often referred to as an ECG or EKG. According to a technique developed by William Einthoven in 1901, points on an ECG are labeled according to a PQRSTU system. The P wave represents activity in the artria and the QRST waves represent ventricular activity. The heart's action is triggered by its own built-in pacing mechanism which comprises a bundle of specialized cardiac muscle fibers known as the sino-atrial node. The P wave represents the time taken for the electrical signal to travel throughout the muscle of the atria, whereas the QRS section represents the ventricular muscle being depolarized and the T section represents the ventricular repolarization. The U section is often not detected and its meaning is not precisely known.
The ECG trace can provide several important pieces of information about the heart. One of the most important measurements to be made from the ECG trace is the PR interval. The PR interval is a measure of the time taken for the electrical impulse to travel through the atria to another specialized muscle bundle which synchronizes the actions of the atria and ventricles. Specifically, the PR interval is a recording of the cardiac impulse traveling to the atrio-ventricular (AV) node and the bundle of His, and then traversing the bundle branches and Purkinje fibers. The PR interval usually lasts 0.12 to 0.21 seconds. A longer period indicates a breakdown in the smooth operation of the AV node. The QRS section should last 0.06 to 0.11 seconds and longer periods typically indicate that the ventricles are acting sluggishly and not getting their electrically impulses simultaneously. The isoelectric ST segment and upright T wave follow the QRS section and represent ventricular repolarization. The ST segment is a sensitive indicator of myocardial ischemia or injury and should be on the isoelectric line.
Arrhythmia is a condition where the heart beats with an irregularity in the force or rhythm. In ventricular tachycardia, there is a rapid and repetitive firing of premature ventricular contractions. When the ventricles contract rapidly with this arrhythmia, the volume of blood ejected into the circulation is often inadequate. This kind of arrhythmia, if left untreated, often degenerates into fatal ventricular flutter or fibrillation. In ventricular fibrillation, there is no recognizable QRS complex and an extremely irregular rhythm. In this type of arrhythmia, virtually no blood is ejected into the systemic circulation, and death will occur if no corrective action is taken.
A major concern during cardiopulmonary bypass procedures is minimizing ischemic damage to the myocardium, thereby avoiding depressed myocardial performance in the post operative period. Prolonged ischemia such as that following myocardial infarction or occurring during long-term coronary bypass procedures causes serious damage to the myocardium. It has been suggested that free radicals are involved in the patho-physiology of ischemia-induced tissue damage. During ischemia, increased reducing equivalents are produced and this may favor the production of O.sub.2.sup.-- anion and other free radical species upon reoxygenation. Many and varied compounds have been reported to reduce the susceptibility of the heart to ischemia/reperfusion injury. These include agents which inhibit free radical production or facilitate their elimination. Other therapeutic agents include calcium channel blockers, prostacyclin analogs and thromboxane inhibitors, sodium channel blockers, and .alpha.- and .beta.-adrenergic receptor blockers. Some of these agents are effective against ischemia and reperfusion induced arrhythmia's, whereas others are effective against only one or the other. Prior to this invention, there were no unique, man-made agents available which abolish arrhythmia's, improve contractility, and protect the heart ultrastructurally.